1. Volume 164, Issue 4, Pages 770-779 (February 2016)
Precision Tumor Recognition by T Cells With Combinatorial Antigen-Sensing Circuits 
Kole T. Roybal, Levi J. Rupp, Leonardo Morsut, Whitney J. Walker, Krista A. McNally, Jason S. Park, Wendell A. Lim 
Cell 
Volume 164, Issue 4, Pages (February 2016)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1
Design of Combinatorial Antigen Sensing Circuits in T Cells Using Sequentially Regulated SynNotch and Chimeric Antigen Receptors
(A) CAR or tumor-specific TCR T cells generally target single antigens, often causing off-target tissue damage. Improved therapeutic T cells will require multiple sensors that recognize combinations of both tumor antigens and tissue-specific antigens, allowing the cells to assess their environment and make more precise decisions on when to activate. Such therapeutic cells would be better equipped to distinguish the target diseased tissue from normal tissue.
(B) New types of receptors that sense combinations of antigens and regulate T cell signaling and transcription must be built to allow for sophisticated cellular decision making and more precise therapeutic T cell responses.
(C) SynNotch receptors are engineered with a custom extracellular ligand-binding domain (e.g., scFv or nanobody) directed toward an antigen of interest (e.g., CD19 or surface GFP). Upon ligand recognition by the synNotch receptor, an orthogonal transcription factor (e.g., TetR-VP64 or Gal4-VP64) is cleaved from the cytoplasmic tail that regulates a custom genetic circuit.
(D) Design of a synNotch AND-gate circuit that requires T cells to sense two antigens to activate. This AND-gate signaling circuit works in two sequential steps: (1) a synNotch receptor allows the T cell to recognize the first antigen A and (2) the T cell expresses a CAR directed toward a second tumor antigen B. If antigen A and antigen B are present, the T cells can activate and kill the target tumor.
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4. Figure 2
SynNotch-Regulated CAR Expression—Combinatorial Antigen Requirement for Jurkat T Cell Activation
(A) Engineering a two-receptor AND-gate circuit: α-CD19 synNotch receptor induces α-mesothelin CAR expression.
(B) Jurkat T cells were engineered with the α-CD19 synNotch tTa receptor and the corresponding response elements controlling α-mesothelin 4-1BBζ CAR expression. The Jurkat T cells must first recognize CD19 on the target tumor via their synNotch receptor in order to initiate CAR expression. After the T cell is primed to activate by CD19, the α-mesothelin CAR can then bind mesothelin and activate the Jurkat cell. Two canonical markers of T cell activation are CD69 upregulation and IL-2 production. The synNotch AND-gate Jurkat T cells should only activate when exposed to target tumor cells expressing both CD19 and mesothelin.
(C) Histograms of the activation marker CD69 in synNotch AND-gate Jurkat T cells co-cultured with single antigen (mesothelin only) or dual antigen (CD19/mesothelin) K562 tumor cells over a 48-hr time course. CD69 was only expressed when the T cells were exposed to dual antigen K562 cells (representative of three independent experiments).
(D) IL-2 ELISA showing IL-2 production by synNotch AND-gate Jurkat cells only when exposed to dual antigen K562 cells (n = 3, error bars are SEM, significance determined by Student’s t test, ∗∗∗∗ = p ≤ ).
(E) Time course of AND-gate T cell activation upon stimulation with dual antigen K562 cells. Expression of the GFP-tagged mesothelin CAR (green) occurs with a half-time of ∼6 hr. Subsequently, activation of the T cell by CAR activation (monitored by CD69 expression) then occurs with a lag of several more hours (t1/2 = ∼13 hr). FACS histograms for CAR expression are shown in Figure S1B.
(F) Time course of AND-gate T cell inactivation upon removal of synNotch ligand. Jurkat T cells expressing the AND-gate circuit were stimulated for 24 hr by plate-bound α-Myc antibody (synNotch receptor has extracellular Myc-tag). START indicates time at which cells were removed from the ligand, and the decay of GFP tagged CAR expression was monitored (t1/2 = ∼8 hr). FACS histograms for CAR expression are shown in Figure S1C.
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5. Figure 3
SynNotch-Regulated CAR Expression in Human Primary T Cells—Combinatorial Antigen Control over Therapeutic T Cell Activation and Tumor Killing
(A) Human primary CD4+ and CD8+ T cells were engineered with the α-GFP nanobody synNotch Gal4VP64 receptor and the corresponding response elements controlling expression of the α-CD19 4-1BBζ CAR. These CD4+ or CD8+ synNotch AND-gate T cells first must sense surface GFP via their synNotch receptor, and only then do they express the α-CD19 CAR and are primed to activate. These AND-gate primary T cells should only activate and produce cytokine or kill target cells if they sense both GFP and CD19.
(B) Primary CD4+ synNotch AND-gate T cells described in (A) were co-cultured with CD19 only or surface-GFP/CD19 K562 cells. Histograms of α-CD19 CAR GFP receptor expression level show that the CAR is only expressed when GFP is present on the surface of the target cell (representative of at least three independent experiments).
(C) The supernatant from CD4+ synNotch AND-gate T cells activated either by CD19 only or GFP/CD19 K562s was analyzed for the presence of 25 cytokines via Luminex. Cytokines were only produced when the T cells were exposed to GFP/CD19 T cells (error bars are SEM, n = 3).
(D) CD8+ synNotch AND-gate primary T cells were engineered as described in (A). As with the CD4+ T cells, the histograms of α-CD19 CAR GFP receptor expression level show that the CAR is only expressed when GFP is present on the surface of the target cell (representative of at least three independent experiments).
(E) Forward and side scatter flow cytometry plots after 24 hr co-culture of CD8+ synNotch AND-gate primary T cells with either CD19 only or GFP/CD19 tumors cells. The T cells fall within the blue gate, and the target CD19 or the GFP/CD19 K562s are in the gray and orange gates, respectively. The synNotch AND-gate T cells only killed the GFP/CD19 K562s, shown by the reduction of cells in the K562 gate (representative of three experiments).
(F) Quantification of replicate CD8+ synNotch AND-gate primary T cell cytotoxicity data shown in panel (E). (n = 3, error bars are SEM, significance determined by Student’s t test, ∗ = p ≤ 0.05). Other examples of synNotch→CAR circuits in primary T cells are shown in Figure S2.
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6. Figure 4
SynNotch Receptors Drive Tumor-Localized CAR Expression In Vivo
(A) Primary human CD4+ and CD8+ T cells were engineered with the α-GFP synNotch Gal4VP64 receptor and the corresponding response elements regulating α-CD19 4-1BBζ CAR IRES effluc expression and were injected i.v. into NSG mice with a Daudi tumor (CD19 only) on the left flank and a surface GFP Daudi (GFP/CD19) tumor on the right flank. Luciferase expression was monitored over 11 days after i.v. injection of engineered T cells.
(B) A representative image of luciferase expression in mice treated as described in (A) at day 7 after T cell injection. Luciferase expression was high in the GFP/CD19 tumor, indicating localized CAR expression only in the dual antigen tumor (n = 2 mice).
(C) Quantification of integrated intensity of luciferase levels in the left-flank Daudi tumor (CD19 only) and surface-GFP Daudi tumor (GFP/CD19) in the right flank. Luciferase expression is enriched in the dual antigen tumor at all time points (error is SD, n = 2).
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7. Figure 5
Selective Combinatorial Antigen Tumor Killing In Vivo by SynNotch-Gated CAR Expression
(A) Primary human CD4+ and CD8+ T cells were engineered with the α-GFP synNotch Gal4VP64 receptor and the corresponding response elements regulating α-CD19 4-1BBζ CAR expression and were injected i.v. into NSG mice with a CD19 K562 tumor on the left flank and a surface-GFP/CD19 K562 tumor on the right flank. Tumor size was monitored over 16 days after i.v. injection of engineered T cells or untransduced T cell controls.
(B) Graphs showing CD19 and GFP/CD19 tumor volumes for mice treated with synNotch AND-gate T cells (top) and untransduced control T cells (bottom). synNotch AND-gate T cells target the dual antigen tumor exclusively and the CD19-only tumor grew at the same rate as in mice treated with untransduced control T cells (n = 5 mice, error bars are SEM, significance determine by Student’s t test, ∗∗ = p ≤ 0.01, ∗∗∗ = p ≤ 0.001).
(C) Tumor volume measurement for individual mice treated with synNotch AND-gate T cells. All mice showed selective killing of the dual antigen tumor.
(D) Kaplan-Meier graphs showing synNotch AND-gate T cells clear GFP/CD19 tumors with 100% of the mice surviving. Mice with CD19-only tumors are not cleared by synNotch AND-gate T cells and have uncontrolled tumor growth. The corresponding tumor growth curves are given on the right of (D) (n = 5 mice, error bars are SEM, significance determine by Student’s t test, ∗∗ = p ≤ 0.01).
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8. Figure 6
SynNotch Receptors Control and Localize CAR T Cell Responses for Precision Immunotherapy
(A) Here, we engineered T cells with synNotch receptors that sense tumor antigens and upregulate expression of a CAR to a second antigen. Thus, these synNotch AND-gate T cells only activate in response to combinatorial antigen recognition in the tumor microenvironment, preventing off-target toxicity mediated by single antigen recognition.
(B) SynNotch AND-gate T cells, unlike therapeutic T cells that target single antigens, can reliably discriminate combinatorial antigen targets from single antigen bystander tissue. Combinatorial antigen sensing by synNotch-CAR T cells could aid in precisely targeting T cells to tumors, preventing off-target toxicity.
(C) synNotch receptors expand the targetable tumor antigen space. Tumor-specific antigens are rare compared to tumor-associated antigens (antigens that are expressed on normal tissue but are more highly expressed on tumors). Since CARs fully activate T cells, resulting in the killing of target tissue, T cells engineered with a single CAR must be targeted to tumor-specific antigens in order to reduce fatal off-target toxicity (upper venn diagram). SynNotch receptors can gate CAR expression and control where the T cells are armed. When targeting tumor-specific antigen combinations, it may now be possible to use CAR receptors directed toward tumor-associated antigens. This should reduce off-target damage to tissues that express the CAR antigen in other parts of the body.
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9. Figure S1
SynNotch-Gated CAR Expression—Combinatorial Antigen Requirement for Jurkat T Cell Activation, Related to Figure 2
(A) α-CD19 synNotch Jurkat T cells controlling expression of the α-mesothelin 4-1BBζ CAR fused to GFP (CD19 synNotch → Meso CAR) were incubated for 48 hr with Meso only or CD19/Meso K562s. Alternatively, the synNotch receptor could be activated by plate-bound anti-Myc antibody (the receptor has an extracellular Myc tag).
(B) Histograms of α-mesothelin CAR GFP expression in synNotch AND-gate Jurkat T cells co-cultured with single antigen (mesothelin only) or dual antigen (CD19/mesothelin) K562 tumor cells over 48 hr time course. CAR expression requires CD19 stimulation, and reaches steady-state with a t1/2 of ∼6 hr.
(C) SynNotch AND-gate Jurkat T cells were stimulated for 24 hr with plate-bound α-myc antibody (binds a Myc-tag on the extracellular domain of the synNotch receptor). After 24 hr, the cells were removed from the α-Myc stimulus and α-mesothelin CAR GFP expression decay was monitored for the subsequent 24 hr.
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10. Figure S2
SynNotch-Gated CAR Expression in Human Primary T Cells—Combinatorial Antigen Control Over Therapeutic T Cell Activation and Tumor Killing, Related to Figure 3
This figure shows other examples of dual antigen circuits: CD19 synNotch→ mesothelin CAR; GFP synNotch→ mesothelin CAR.
(A) CD4+ primary T cells were engineered with the α-CD19 synNotch Gal4VP64 receptor and the corresponding response elements controlling α-mesothelin 4-1BBζ CAR GFP expression. The T cells were then co-cultured with mesothelin only, CD19 only, or CD19/mesothelin K562s for 24 hr and CD69 upregulation and IL-2 production were assayed.
(B) Histograms showing α-mesothelin CAR GFP levels and CD69 levels on CD4+ synNotch primary T cells cultured as described in (A). The α-mesothelin CAR was only expressed when CD19 was on the target K562s and the T cells only expressed the activation marker CD69 when both CD19 and mesothelin were on the target K562s (representative of 3 experiments).
(C) IL-2 levels from supernatant harvested from cultures described in panel (A). IL-2 was only produced when the T cells were exposed to target cells expressing both CD19 and mesothelin (n = 3, error bars are SEM, significance determined by Student’s t test ∗∗∗ = p ≤ 0.001).
(D) CD8+ primary human T cells were engineered as described in panel (A). For CD8+ T cells specific cytotoxicity of mesothelin only, CD19 only, or CD19/mesothelin target K562s was determined. The synNotch AND-gate CD8+ T cells should only kill dual positive K562s.
(E) Histograms showing α-mesothelin CAR EGFP levels on CD8+ synNotch primary T cells cultured as described in panel (A). The α-mesothelin CAR was only expressed when CD19 was on the target K562s (representative of 3 experiments).
(F) Quantification of replicate CD8+ synNotch AND-gate primary T cell cytotoxicity showing specific killing of target K562s with both CD19 and mesothelin expression (n = 3, error bars are SEM, ∗∗∗ = p ≤ 0.001).
(G) CD4+ primary T cells were engineered with the α-GFP nanobody synNotch Gal4VP64 receptor and the corresponding response elements controlling α-mesothelin 4-1BBζ CAR GFP expression. The T cells were then co-cultured with mesothelin only, GFP only, or GFP/mesothelin K562s for 24 hr and CD69 upregulation and IL-2 production were assayed.
(H) Histograms showing α-mesothelin CAR GFP levels and CD69 levels on CD4+ synNotch primary T cells cultured as described in (G). The α-mesothelin CAR was only expressed when GFP was on the target K562s and the T cells only expressed the activation marker CD69 when both GFP and mesothelin were on the target K562s (representative of 3 experiments).
(I) IL-2 levels from supernatant harvested from cultures described in (G). IL-2 was only produced when the T cells were exposed to target cells expressing both GFP and mesothelin (n = 3, error bars are SEM, ∗∗∗∗ = p ≤ ).
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11. Figure S3
SynNotch Receptors Drive Tumor-Localized CAR Expression In Vivo, Related to Figure 4
(A) Representative dot plots showing expression of the α-GFP synNotch Gal4VP64 receptor and the corresponding response elements regulating α-CD19 4-1BBζ CAR IRES effluc in primary CD4+ and CD8+ T cells. The T cells outlined by the red box were sorted and used for in vivo and in vitro experiments.
(B) Bar graph showing luciferase activity in synNotch AND CD4+ and CD8+ T cells from (A) after exposure for 24 hr with GFP- or GFP+ Daudi cells. Luciferase was specifically expressed in response to GFP (n = 3, error bars are SEM, ∗∗∗∗ = p ≤ ).
(C) Tumor growth curves are given for mice analyzed in Figure 4C.
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12. Figure S4
Selective Combinatorial Antigen Tumor Killing In Vivo by SynNotch-Gated CAR Expression, Related to Figure 5
(A) Representative dot plots showing the expression of the α-GFP synNotch Gal4VP64 receptor and the corresponding response elements regulating α-CD19 4-1BBζ CAR in primary human CD4+ and CD8+ T cells. T cells in the red-boxed quadrant were sorted and used for experiments in Figure 5.
(B) Flow cytometry plots showing the expression level of CD19 and GFP (green) on dual antigen K562s and CD19 on single antigen K562s (purple) utilized for in vitro and in vivo experiments.
(C) Tumor growth curves for individual mice with bilateral CD19 (left flank) and GFP and CD19 (right flank) tumors treated with control untransduced CD4+ and CD8+ T cells. The data underlie Figure 5B lower panel.
(D) Primary human CD4+ and CD8+ T cells were engineered with the α-GFP synNotch Gal4VP64 receptor and the corresponding response elements regulating α-CD19 4-1BBζ CAR expression and were injected i.v. into mice with a CD19 K562s on the left flank and a surface-GFP K562 tumor on the right flank to test if the T cells migrate from the GFP only ‘priming tumor’ and kill the off-target CD19 only tumor. Tumor size was monitored over 16 days after i.v. injection of engineered T cells or untransduced T cell controls.
(E) Graph showing CD19 tumor volumes for mice treated with synNotch AND-gate T cells (solid line) or untransduced control T cells (dotted line). The CD19 tumor is not targeted, suggesting there is no migration of primed T cells from the GFP only tumor (n = 5, error bars are SEM, no significant difference at any time points based on Student’s t test, p > 0.05).
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